It is widely accepted that adenosine plays an important role in the regulation of coronary blood flow. The cellular and subcellular mechanisms by which adenosine produces the relaxation of coronary smooth muscle have been investigated in this laboratory for the last several years but are still not completely understood. Several possibilities, including the existence of adenosine receptor (Ra subtype) from this laboratory, changes in the intracellular calcium and ion fluxes have been proposed but the direct evidence is lacking. The proposed studies which are the continuation of the earlier work are directed at some of these possibilities. A combination of models will be used for these studies. We propose to study the: (a) relaxation of precontracted coronary artery by adenosine and its analogs in the absence of calcium and its antagonism to define the receptor subtype; (b) intracellular calcium lowering effect of adenosine and its analogs by measuring the efflux of calcium in the absence of extracellular calcium and the direct effects on intracellular organelles using skinned muscle; (c) intracellular calcium lowering effect of adenosine and its analogs using 3, 4, diaminopyridine induced phasic contraction of the coronary artery and its comparison to calcium entry blockers; (d) effect of adenosine and its analogs on 42K, 24Na and 36C1 fluxes; (e) binding of 3H-NECA (Ra site) and 125I-HPIA (Ri site) in purified plasma membrane, coronary microvessels and isolated cells (vascular smooth, cardiac muscle, endothelial); (f) order of potency of the adenylate cyclase activation using adenosine analogs and the protein phosphorylation in coronary plasma membranes; (g) characterization of adenosine receptor in human coronary arteries; (h) isolation and purification of adneosine receptor binding protein from coronary plasma membrane fraction; (i) adenosine receptor in hypertensive rat aortae both in the presence and absence of calcium using isolated vascular rings and measure the adenosine receptor binding in coronary microvessel preparation of hypertensive rat hearts; and finally (j) release of adenosine in the presence of methylxanthine during reactive hyperemia and adenosine infusion using Langendorf hearts. The data obtained from these studies should represent a major advance in our understanding of the metabolic regulation of coronary flow at a cellular and subcellular level which can lead to the development of rationale therapeutic approaches for regional blood flow disorders.